Protein designated PEG modification method and obtained PEG modified protein

A protein and fixed-point technology, applied in the field of protein fixed-point PEG modification, can solve the problems of destroying the biological activity of proteins, uneven modified products, unstable curative effect, etc., and achieve the effects of avoiding by-products, low cost and simple operation

Inactive Publication Date: 2014-11-05
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because multiple groups on the protein molecule can react with polyethylene glycol, some side reactions will occur during the chemical modification of the protein with polyethylene glycol, resulting in a complex mixture of multiple PEG-modified proteins, and It is possible to destroy the biological activity of the modified protein, and eventually lead to inhomogeneous modified products, many by-products, and unstable curative effect, which limits the application of PEG in protein modification

Method used

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  • Protein designated PEG modification method and obtained PEG modified protein
  • Protein designated PEG modification method and obtained PEG modified protein
  • Protein designated PEG modification method and obtained PEG modified protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0128] Example 1 Preparation of Recombinant Human Growth Hormone

[0129] (1) Obtaining the recombinant vector containing human growth hormone gene:

[0130] Source of recombinant gene:

[0131] Recombinant human growth hormone gene, i.e. hGH gene: According to the human growth hormone gene published by PubMed, gene synthesis was carried out in Shanghai Bioengineering Company, that is, the gene of human growth hormone was obtained, i.e. hGH gene, which has such as SEQ ID NO: 17 Nucleotide sequence shown.

[0132]pMAL-c5x expression vector: derived from New England BioLabs Inc. Cat. NO. N8108s. The expression vector contains the MBP gene (i.e. the maltose binding protein gene) and the gene of the FXa recognition peptide IEGR. When the foreign gene is inserted, the N-terminus of the resulting fusion protein is fused with the MBP protein and the IEGR amino acid sequence after induced expression; when the recombinant protein has When the MBP protein is tagged, it is beneficial ...

Embodiment 2

[0203] Example 2 Comparison of the ability of sortase to modify different fusion human growth hormones

[0204] The source of the sortase SrtA (i.e. Sortase A): according to the construction of the Sortase A recombinant expression vector recorded in "The Inhibitory Effect of Rutin on Staphylococcus aureus Sortase A" published by Wang Yanan et al. in the Journal of Jilin Agricultural University in 2013 and It was prepared by the expression and purification method, and the obtained sorting enzyme SrtA was detected by SDS-PAGE, and the detection result was as follows: Figure 12 , where M is the molecular weight marker; lane 1 is the purified sortase; band T is the sortase SrtA; after detection, the concentration of the sortase SrtA is 5 μmol / L.

[0205] Take the primary products prepared from recombinant strain 1 to recombinant strain 7 in Example 1 (i.e. collect the bacterial cells, centrifuge the supernatant obtained after freezing and thawing), carry out PEG modification, com...

Embodiment 3

[0229] Example 3 PEG modification of growth hormone using sortase

[0230] Take the recombinant human growth hormone 1 prepared in Example 1, and add the sortase SrtA (Example Prepared by the preparation method provided in 2, the concentration is 5 μmol / L), polyethylene glycol modified dipolyglycine (PEG modifier, derived from Qiangyue Biotechnology Co., Ltd., which has the structure shown in formula (I)) , after mixing evenly, adjust the pH value to 8, and react for 18 hours at 25°C;

[0231]

[0232] Where m=4; n=220, the corresponding molecular weight of PEG is 10K.

[0233] Using the HPLC method, the resulting modified product was separated, and the eluted fraction was collected to obtain PEG-modified human growth hormone 1. The results were as follows: Figure 14 Shown, where peak 1 represents 10K PEG-modified human growth hormone 1, that is, PEG-modified human growth hormone 1; peak 2 represents unmodified recombinant human growth hormone 1; where numbers 1-21 refer...

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Abstract

The invention belongs to the technical field of biological engineering, and discloses a protein designated PEG modification method and an obtained PEG modified protein. The protein designated PEG modification method of the invention is as below: acquiring a protein, fusing a sorting motif recognized by a sortase at the C terminal of the protein to obtain a recombinant protein; mixing a PEG modifier, the recombinant protein and sortase, conducting peptide bond cutting and connection reaction to obtain a PEG modified protein, wherein the PEG modifier has a structure shown in a formula (I). The invention utilizes the sortase to realize designated PEG modification at the C terminal of the protein; the whole operation is simple and low in cost, avoids multisite modification in protein PEG modification, is more conducive to application of PEG modification in protein modification; m represents the degree of polymerization, and satisfies the relation of 1<=m<= 5; and n represents the degree of polymerization, and satisfies the relation of 110<= n<=1100.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for protein site-specific PEG modification and the resulting PEG-modified protein. Background technique [0002] Polyethylene glycol (PEG) is a polymer of the hydrolysis product of ethylene oxide, which is a hydrophilic, uncharged linear macromolecule. Covalently linking hydrophilic polymer polyethylene glycol is a method to increase the water solubility of bioactive molecules (including proteins, peptides, especially hydrophobic biomolecules), improve their bioavailability, prolong serum half-life, and regulate immunogenicity , Improve biological activity commonly used methods. Among them, the use of PEG to modify proteins to prolong their half-life, reduce immunogenicity, and increase stability is a technology that has been successfully applied. This technology has been widely used in the modification of protein drugs, for example, PEG-asparaginase...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/107C07K14/61A61K38/27A61K47/48A61P5/06A61P13/12A61P31/18
Inventor 王大成焦虎平李文亮李云辉杨秀云邓旭明房恒通李占军吕岩张英
Owner JILIN UNIV
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